practicals Flashcards
how can you tell the difference between a digested and undigested sample on a gel?
undigested DNA is represented by a sharp band, smearing represents digested DNA
when should you disconnect the electrode from the power supply when running a gel?
when the dye has moved 2/3 of the way
what is the role of ethidium bromide in an electrophoresis gel?
fluorescent tag
what is a polyanion?
a molecule or chemical complex which has negative charges at several sites
what is the role of EDTA in stop solution?
chelates Mg2+
how do you calculate the length in base pairs?
M(Da)/650
what is the beer lambert law? (rearranged)
c=A/molar coefficient x L
name the techniques that can be used to determine the catalytic activity of an enzyme
spectrophotometry, isotopic labelling, titration, quantitative chemical analysis
what are the advantages of spectrophotometric techniques?
reactions can be followed continuously
rapid
low concentrations measurable
what does the x-intercept represent on a lineweaver-burk plot?
-1/Km
what does the y-intercept represent on a lineweaver-burk plot?
1/Vmax
what effect does a non-competitive inhibitor have on Vmax and Km?
Vmax = reduced Km= same
what effect does a competitive inhibitor have on Vmax and Km?
Vmax= same Km= increased
what does Km effectively measure?
affinity of an enzyme for the substrate
what is the limitation of lineweaver-burk plots?
they lay undue emphasis on data obtained at low substrate concentrations
how is activity of B-Galactosidase measured?
measuring ONP production by hydrolysis of the artificial substrate ONPG
why is turkey blood used when purifying histone proteins?
turkey RBCs are nucleate, mammalian RBCs are not
what is AEBSF?
a water soluble protease inhibitor
what is Triton X-100?
detergent used to lyse cells so histones can be extracted
why is micrococcal nuclease digestion carried out when purifying histone proteins?
targets nucleic acids not histones
How does SDS allow proteins to be separated by mass?
creates denaturing conditions that separate proteins by molecular weight
which technique is used to separate histone proteins?
electrophoresis through a polyacrylamide gel
what is the first step when separating histone proteins?
incubation and washing in buffer to break cell membrane and denature proteins not associated with histones
what is the role of MNase digestion in the separation of histones?
digests insoluble chromatin into insoluble oligonucleosomes
name two factors which affect the rate at which proteins move through a polyacrylamide gel
molecular weight and the presence of charged side chains
